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Preparation method of alpha-glucan antigen and specificity monoclonal antibody

A monoclonal antibody, glucan technology, applied in the preparation methods of peptides, chemical instruments and methods, anti-animal/human immunoglobulins, etc., can solve the problems of low antibody titer and weak antigen immunogenicity, etc. Achieve high immunogenicity and high potency

Inactive Publication Date: 2011-07-20
GUANGZHOU SUGARCANE IND RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In CN 101139395A, a method for preparing an α-glucan monoclonal antibody is disclosed, by combining α-glucan with keyhole limpet hemoglobin and bovine The serum protein was coupled to obtain the antigen, and then the monoclonal antibody was prepared according to the conventional method, but the titer of the obtained antibody was extremely low, and the titer ratio did not exceed 10-4, and it could not interact with α-glucan at all. The sugar reacts effectively and gets precipitated, the main reason is the weak immunogenicity of the antigen

Method used

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  • Preparation method of alpha-glucan antigen and specificity monoclonal antibody
  • Preparation method of alpha-glucan antigen and specificity monoclonal antibody
  • Preparation method of alpha-glucan antigen and specificity monoclonal antibody

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Experimental program
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Effect test

preparation Embodiment 1

[0038] 1) Mix Dextran 40 (molecular weight 40kDa) and BSA at a mass ratio of 1:1, then dissolve in distilled water, adjust the pH of the solution to 6, and the solid content is 6% (w / v), freeze-dry, and grind into powder ;

[0039]2) Put the powder at a constant temperature of 60°C, maintain a relative humidity of 79%, and react for 7 days to obtain a Dextran-BSA cross-linked product, which is the α-glucan antigen.

[0040] The Dextran-OVA cross-linked product was prepared under the same conditions.

[0041] Samples were taken at different reaction times, and the products were subjected to SDS-PAGE electrophoresis and stained with sugar staining and Coomassie brilliant blue R250. The results were as follows: figure 1 as shown, figure 1 a is sugar stained, figure 1 b is stained with Coomassie brilliant blue R250. figure 1 Among them, band 1 is BSA; 2 is Dextran 40-BSA coupling reaction 0d; 3 is Dextran 40-BSA coupling reaction 1d; 4 is Dextran 40-BSA coupling reaction 3d; 5...

preparation Embodiment 2

[0044] 1) Mix Dextran 400 (molecular weight 400kDa) and BSA at a mass ratio of 2:1, then dissolve in distilled water, adjust the pH of the solution to 5, and the solid content is 8% (w / v), freeze-dry, and grind into powder ;

[0045] 2) Put the powder at a constant temperature of 50°C, keep the relative humidity at 90%, and react for 3 days to obtain a Dextran-BSA cross-linked product, which is the α-glucan antigen.

[0046] The Dextran-OVA cross-linked product was prepared under the same conditions.

preparation Embodiment 3

[0048] 1) Mix Dextran 1000 (molecular weight 1000kDa) and BSA at a mass ratio of 1:2, then dissolve in distilled water, adjust the pH of the solution to 8, and solid content to 6% (w / v), freeze-dry, and grind into powder ;

[0049] 2) Put the powder at a constant temperature of 55°C, maintain a relative humidity of 60%, and react for 5 days to obtain a Dextran-BSA cross-linked product, which is the α-glucan antigen.

[0050] The Dextran-OVA cross-linked product was prepared under the same conditions.

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Abstract

The invention discloses a preparation method of an alpha-glucan antigen and a specificity monoclonal antibody. The preparation method of the antigen comprises the following steps: mixing alpha-glucan and protein; dissolving with water; regulating the pH of solution to 5-8; carrying out freeze drying, and grinding into powder; putting the powder in the environment with constant temperature of 50-60DEG C and relative humidity of 60-90%; reacting for at least three days to obtain the alpha-glucan antigen. The preparation method of the antibody comprises the following steps: mixing the prepared antigen with an adjuvant, emulsifying the mixture, and injecting the emulsified mixture into animal bodies; blending the splenocyte and oncocyte of the animal body with the high serum valence to obtain a hybridoma cell capable of secreting the antibody; and producing the antibody by the hybridoma cell or with an ascites method. The antibody prepared by the method has high immunogenicity, and can be used for effectively inducing to generate immune response to obtain the antibody with the excellent property. The antibody obtained by the method provided by the invention can be subjected to specificity reaction with the alpha-glucan to generate macroscopic precipitates, and the antibody has high valence and can be used for the quantitative analysis on the alpha-glucan.

Description

technical field [0001] The invention relates to a preparation method of alpha-glucan antigen and specific monoclonal antibody. Background technique [0002] α-glucan (Dextran) is a polysaccharide composed entirely of glucose monomers, molecular formula (C 6 h 10 o 5 ) n , the molecular weight is generally between tens of thousands to several million, usually white amorphous powder solid, sticky, viscosity increases with the increase of molecular weight and concentration, odorless and tasteless, easily soluble in water, solubility increases with molecular weight increase and decrease. It can exist stably in normal temperature and neutral solution, and it will decompose when encountering strong acid, and its end group will be easily oxidized in alkaline solution, and it can gradually change color or decompose when heated. [0003] The harm of α-glucan to the sugar industry has the following aspects: (1) causing sugar loss and reducing the recovery rate; (2) increasing the...

Claims

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Application Information

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IPC IPC(8): C07K1/113C07K14/765C07K16/18
Inventor 梁达奉颜江华曾练强蚁细苗李雨虹王生育张远平黄玉南钟映萍
Owner GUANGZHOU SUGARCANE IND RES INST
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